U.S. patent number 6,099,804 [Application Number 08/950,362] was granted by the patent office on 2000-08-08 for sensor and membrane for a sensor.
This patent grant is currently assigned to Radiometer Medical A/S. Invention is credited to Allan Milton Byrnard, Lydia Dahl Clausen, Jesper Svenning Kristensen.
United States Patent |
6,099,804 |
Clausen , et al. |
August 8, 2000 |
Sensor and membrane for a sensor
Abstract
In a sensor for measuring an analyte in a biological sample
whose measuring surface is covered by or comprises a membrane, the
membrane has free groups on the surface facing the sample. The
surface is modified such that a hydrophilic component is
immobilized on the surface in such a manner that chains of the
hydrophilic component are chemically bonded to free groups on the
surface. Thus, the surface is provided with a more hydrophilic
character relative to its unmodified state.
Inventors: |
Clausen; Lydia Dahl (Lynge,
DK), Byrnard; Allan Milton (Copenhagen S,
DK), Kristensen; Jesper Svenning (Lyngby,
DK) |
Assignee: |
Radiometer Medical A/S (Br.o
slashed.nsh.o slashed.j, DK)
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Family
ID: |
8092643 |
Appl.
No.: |
08/950,362 |
Filed: |
October 6, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCTDK9700111 |
Mar 14, 1997 |
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Foreign Application Priority Data
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Mar 29, 1996 [DK] |
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0360/96 |
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Current U.S.
Class: |
204/403.09;
204/403.11; 204/416; 210/490; 210/500.21; 210/500.24; 210/500.27;
210/500.36; 422/82.01; 422/82.03 |
Current CPC
Class: |
C12Q
1/002 (20130101); B01D 67/0093 (20130101) |
Current International
Class: |
B01D
67/00 (20060101); C12Q 1/00 (20060101); G01N
027/00 () |
Field of
Search: |
;204/194,400,403,416
;435/287.9 ;436/95 ;422/82.01
;210/490,500.21,500.24,500.27,500.36 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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4002513 C1 |
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Jul 1991 |
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DE |
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WO 92/04438 |
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Mar 1992 |
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WO |
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WO 96/18498 |
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Jun 1996 |
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WO |
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WO 96/17883 |
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Jun 1996 |
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WO |
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Other References
W Matuszewski et al, "Amperometric Glucose Biosensor for an
Undiluted Whole-Blood Analysis", Anal. Sci., Jun. 1994 vol. 10, pp.
423-428..
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Primary Examiner: Redding; David A.
Attorney, Agent or Firm: Waddell; Mark E. Haracz; Stephen M.
Bryan Cave LLP
Parent Case Text
This is a continuation of International Application No.
PCT/DK97/00111.
Claims
What is claimed is:
1. A sensor for measuring an analyte in a biological sample
comprising a measuring surface having an associated membrane, said
membrane comprising a modified membrane surface facing the sample
through which the analyte to
be measured permeates toward the measuring surface, said membrane
surface having free groups prior to surface modification, wherein
the surface is modified by immobilizing a hydrophilic component on
the surface in such a manner that chains of the hydrophilic
component are chemically bonded to the free groups on the surface
forming said modified membrane surface, thus providing said
modified membrane surface with a more hydrophilic character
relative to its unmodified state.
2. The sensor according to claim 1, wherein said membrane has pores
extending from the first surface of said membrane to the opposite
surface of said membrane.
3. The sensor according to claim 2, wherein chains of the
hydrophilic component have penetrated into the pores of the
modified membrane and are bonded to free groups on the surface of
the pores.
4. The sensor according to claim 1, wherein said membrane is a
multi-layered membrane and wherein the layer which faces the sample
has pores which extend from the first surface of the layer to the
opposite surface of the layer.
5. The sensor according to claim 4, wherein chains of the
hydrophilic component have penetrated into the pores of said
modified membrane and are bonded to free groups on the surface of
the pores.
6. The sensor according to claim 4, wherein said membrane surface
facing the sample comprises polyethylene terephtalate.
7. The sensor according to claim 1, wherein said hydrophilic
component is polyethylene glycol.
8. The sensor according to claim 7, wherein chains of polyethylene
glycol are bonded to free --COOH groups of said membrane
surface.
9. The sensor according to claim 1 wherein the chemical bond is an
amide bond.
10. The sensor according to claim 1 wherein the chemical bond is an
ester bond.
11. The sensor according to claim 1 wherein the hydrophilic
component has at least two hydrophilic groups.
12. The sensor according to claim 11 where the hydrophilic
component has two hydrophilic groups.
13. The membrane for use in a sensor comprising a modified membrane
surface having free groups prior to surface modification, wherein
the surface is modified by immobilizing a hydrophilic component on
the surface in such a manner that chains of the hydrophilic
component are chemically bonded to the free groups on the surface
forming the modified membrane surface, thus providing the modified
membrane surface with a more hydrophilic character relative to its
unmodified state.
14. The membrane according to claim 13 wherein the membrane has
pores extending from the first surface of the membrane to the
opposite surface of the membrane.
15. The membrane according to claim 13 wherein the membrane is a
multi-layered membrane and wherein the layer which faces the sample
has pores which extend from the first surface of the layer to the
opposite surface of the layer.
16. The membrane according to claim 14 wherein chains of the
hydrophilic component have penetrated into the pores of the
modified membrane and are bonded to free groups on the surface of
the pores.
17. The membrane according to claim 15 wherein chains of the
hydrophilic component have penetrated into the pores of the
modified membrane and are bonded to free groups on the surface of
the pores.
18. The membrane according to claim 13 wherein the modified
membrane comprises polyethylene terephtalate.
19. The membrane according to claim 13 wherein the hydrophilic
component is polyethylene glycol.
20. The membrane according to claim 13 wherein chains of
polyethylene glycol are bonded to free COOH groups of the membrane
surface.
21. The membrane according to claim 13 wherein the membrane is kept
in a stretched position by a primarily circular membrane ring.
22. The membrane according to claim 13 wherein the membrane ring is
connected with a jacket adapted to be mounted on an electrode.
23. The membrane according to claim 13 wherein the chemical bond is
an amide bond.
24. The membrane according to claim 13 wherein the chemical bond is
an ester bond.
25. The membrane according to claim 13 wherein the hydrophilic
component has at least two hydrophilic groups.
26. The membrane according to claim 25 wherein the hydrophilic
component has two hydrophilic groups.
Description
The invention relates to a sensor for measuring an analyte in a
biological sample having a measuring surface covered by or
comprising a membrane through which the analyte to be measured
penetrates towards the measuring surface, said membrane having free
groups on the surface facing the sample.
One of the difficulties in selecting a membrane for a sensor to be
used for measuring on whole blood is that some membrane materials
have a surface character which tends to make some macromolecular
elements of the blood, such as blood proteins and red blood cells,
stick to the surface of the membrane. This results in a poor
measurement quality of the sensor as reproducible measurement
results are not obtainable when the measuring surface is coated.
The tendency towards surface coating seems to depend on i.a.
whether the membrane surface has a hydrophilic or hydrophobic
character as a hydrophobic surface has a stronger tendency towards
coating.
It has been attempted to solve the problem by making the membrane
facing the sample or outer membrane of materials having minimal
tendency towards coating. An analysis of different outer membranes
applied in a biosensor for measuring glucose is disclosed in
Matuszewski W, Trojanowicz M, Lewenstam A, Moszcyska and
Lange-Moroz E. Amperometric glucose biosensor for an undiluted
whole-blood analysis. Analytical Sciences 1994; Vol. 10: 423-428.
The article discloses the application of e.g. polycarbonate,
polyester, polyethylene terephthalate, polypropylene, etc.
According to the article, good results are obtained by using an
outer membrane of polypropylene (PP) from Celanese. The surface
character of said membrane is usually hydrophobic and consequently
should not be very suitable as an outer membrane, cf. above.
However, wetting the membrane with a hydrophilic
component/surfactant provides a hydrophilic surface character, and
good results have been obtained. The article discloses wetting with
different hydrophilic components/surfactants. The best result was
obtained by wetting a PP membrane with a surfactant designated
Triton X-100.
The wetting process proper consisted in dipping the PP membrane
into a solution of surfactant for 10 minutes, rinsing the membrane
with distilled water followed by wiping the membrane.
Even after a number of measurements on whole blood, the outer
membrane showed no signs of adsorbing red blood cells. Further,
within a two-week period no significant change of the measuring
signal occurred. In between the measurements the outer membrane was
kept in a phosphate buffer solution at 4.degree. C.
However, the sensor disclosed in the article has the disadvantage
that it is not suitable in automatic analyzers such as the analyzer
ABL.TM.620 from Radiometer Medical A/S, Copenhagen, Denmark because
the surface treatment of the outer membrane cannot stand the
repeated rinse procedures which the analyzer performs automatically
(more specifically an instrument-driven rinse procedure upon each
measurement). These procedures are performed by means of special
rinse solutions. The hydrophilic component/surfactant will be
washed off and the outer membrane will gradually loose its
hydrophilic character. This will result in coatings on the surface
of the membrane causing poor reproducibility and/or decreasing
measuring signals, cf. above.
Accordingly, the purpose of the invention is to provide a sensor
with a membrane facing the sample which has no tendency towards
coating and which is resistant to repeated rinse procedures.
This is obtained by the sensor according to the invention which is
characterized in that the surface is modified such that a
hydrophilic component is immobilised on the surface in such a
manner that chains of the hydrophilic component are chemically
bonded to free groups on the surface, thus providing the surface
with a more hydrophilic character in relation to its unmodified
state.
As the hydrophilic component is chemically bonded to the free
groups on the surface of the membrane, the component is not leached
out, even after repeated rinse procedures of the sensor. Further,
the sensor requires no special storage in between the measurements
as the membrane with the chemically bonded hydrophilic component is
more stable than e.g. a membrane only wetted with a hydrophilic
component.
The sensor according to the invention may be any sensor whose
measuring
surface is covered by or comprises a sample-contacting membrane. It
is most likely that all sensors applied for measuring on biological
samples may advantageously be embodied according to the invention.
Examples of such sensors are gas sensors such as sensors for
measurement of oxygen, carbon dioxide and ammonia; electrolyte
sensors such as sensors for measurement of lithium, potassium,
sodium, magnesium, calcium, ammonium, bicarbonate and chloride;
biosensors such as enzyme sensors for measurement of glucose,
cholesterol, lactate, creatinin, urea, carbamide, pyruvate,
alcohol, bilirubin, ascorbate, phosphate, protein, triglycerides,
phenylananine, tyrosin and hypoxanthine and immunosensors.
Sensors according to the invention may be based on any suitable
measuring technique and may thus be based on e.g. electrochemical
or optical principles.
The sensor according to the invention is primarily an
electrochemical (amperometric) enzyme electrode having a two- or
multi-layered membrane where the membrane layer facing the sample
is porous. The porosity of the membrane layer facing the sample is
primarily selected such that the membrane layer is a so-called
substrate-limiting membrane layer as further disclosed in e.g. the
specification of Danish Patent No. DK 170103.
In this context, a biological sample is a sample of whole blood,
serum, plasma, or a sample of a body fluid in a natural or treated
state.
The material for the membrane layer facing the sample (outer
membrane layer) may be selected among all materials suitable for
sensors and is most often a polymeric foil. The particular
selection may take place in the light of requirements as to the
permeability of certain agents, requirements as to resistance,
strength, etc. It is merely required that the membrane has free
groups on its surface to which the hydrophilic component may be
bonded. Said free groups may be present in the natural state of the
membrane or be applied by means of e.g. basic hydrolysis or plasma
treatment.
Different rinse conditions may have an impact on the selection of a
hydrophilic component. The hydrophilic component must be adapted so
that sufficient washing from the pores of the membrane of e.g. the
blood components, penetrating into the pores, may be obtained in
between the measurements. Adaption of the molecular weight of the
hydrophilic component to different rinse conditions of the analyzer
may also be required in order to obtain sufficient washing of the
pores in between the measurements.
A polyethylene glycol (PEG) of suitable chain length/molecular
weight and heparin are particularly preferred hydrophilic
components which may be bonded to free groups of the membrane or
membrane portion, e.g. free groups of carboxylic acid with a
suitable coupling reagent. Other suitable hydrophilic components or
surface modified agents may be selected among hydrophilic natural
polymers having similar surface modifying properties as PEG, e.g.
hyaluronic acid, phospholipides, agarose, chitosan, cyclodextrin,
alginate, collagen, lignin, pectin and polysaccharides and
celulose-based polymers such as dextrin, hydroxyalkyl celluloses,
cellulose acetates, albumin, gelatin, agar, carageenans and starch;
hydrophilic synthetic polymers having similar surface modifying
properties as PEG, e.g. polyvinylalcohol/polyvinylacetates,
polyvinyl pyrrolidone, hydroxymethylmethacrylate,
hydroxyethylacrylate, acrylic acid, allyl alcohol and acrylic
polymers (hydrogels); plasma polymerised polymers (acryls).
Besides, reconcilability with the sensor is, of course,
presumed.
It is important that the chains of the hydrophilic component are
not so long that shear forces detach them from the surface of the
membrane.
It is also important in connection with porous membranes or
membrane layers that the chains of the hydrophilic component are
not so long that they block the pores, thus preventing the analyte
to permeate through the membrane or the membrane layer.
In this context, the unmodified state of the surface of the
membrane is the state prior to modification with the hydrophilic
component. This need not necessarily be the natural state in that
some membranes, as mentioned above, may be subjected to basic
hydrolysis or plasma treatment in order to apply free groups on the
surface. In this case, the unmodified state is the state after
application of the free groups, but prior to the modification with
the hydrophilic component.
In a preferred embodiment, the membrane or membrane layer is porous
and chains of the hydrophilic component are penetrated into the
pores of the membrane and bonded to free groups of the surfaces of
the pores. In this case, when selecting a hydrophilic component it
is furthermore important to ensure that the chains are not so long
that they cannot get into the pores of the membrane or membrane
layer and when placed on the surfaces of the pores will not block
the pores by reducing the open cross-sectional area of the pores
too much.
BRIEF DESCRIPTION OF DRAWINGS
In the following the invention is further explained with reference
to the drawing, in which
FIG. 1 is a sectional view of a sensor according to the
invention,
FIG. 1a is an enlarged section of FIG. 1,
FIG. 2 is a schematic view of a section of an outer membrane for a
sensor according to the invention prior to surface modification,
and
FIG. 3 is a schematic view of an outer membrane for a sensor
according to the invention after surface modification.
The sensor of FIG. 1 is a sensor for measuring glucose. The
measuring technique of the sensor is well-known. It is based on
enzymatic conversion of glucose and oxygen into hydrogen peroxide
(H.sub.2 O.sub.2) and glyconic acid. Subsequently, the produced
H.sub.2 O.sub.2 is detected by an amperometric electrode. The
sensor 1 is adapted to be placed in an analyzer for measurement of
a blood sample such as the above-mentioned analyzer ABL.TM.620 from
Radiometer Medical A/S, Copenhagen, Denmark.
The sensor 1 primarily comprises an electrode 2 mounted on a
membrane ring 3. The electrode 2 comprises a Pt anode 4 connected
with a Pt wire 5 which is connected with anode contact 7 of silver
through a micro plug 6. The Pt anode 4 and a part of the Pt wire 5
are glued into a glass part 8. Between the glass part 8 and the
micro plug 6 the Pt wire 5 is protected by a tube 9 of heat shrink
tubing. A tubular reference electrode 10 of silver surrounds the
upper part of the glass part 8 and extends throughout the electrode
2's length to the anode contact 7 which is secured inside the
reference electrode 10 by means of a fixation device 11 and epoxy
12. The lower part of the glass part 8 is surrounded by an
electrode support 13 at which the membrane ring 3 is placed. The
upper part of the reference electrode 10 is surrounded by a plug
part 14, serving to mount the electrode 2 in a matching plug on an
analyzer (not shown) and to securing a jacket 15. Between the
electrode 2 and the jacket 15 are placed gaskets 16 and 17,
ensuring that any electrolyte placed at the measuring surface of
the electrode 2 does not evaporate from the sensor 1.
The membrane ring 3, which is mounted at one end of the jacket 15,
comprises a ring 20. A membrane 21 is kept in a stretched position
above the lower opening of the ring 20. Said membrane 21, which is
more clearly shown in FIG. 1a, consists of an approx. 6 .mu.m
porous membrane layer 22 of cellulose acetate (CA) onto which is
applied an approx. 1 .mu.m enzyme layer 23 of a cross-linked
glucose oxidase (5 units per membrane) onto which is further
applied an approx. 12 .mu.m porous membrane layer 24 of
polyethylene terepthalate (PETP). The membrane 21 is placed such
that the CA membrane layer 22 faces the Pt anode 4 of the electrode
2 when the membrane ring 3 is mounted.
The PETP membrane layer 24 serves as a diffusion-limiting membrane.
It ensures that the amount of glucose penetrating from the sample
through the glucose oxidase layer 23, where the enzymatic
conversion takes place, is not larger than sufficient oxygen for
the conversion is available all the time. An example of how to
prepare a PETP membrane layer is set forth below.
After the conversion in the glucose oxidase layer 23 the H.sub.2
O.sub.2 formed penetrates through the CA membrane layer 22 to the
Pt anode 4 where it is oxidized and thereby detected. The Pt anode
4 is polarized to +675 mV with an Ag/AgCl reference electrode.
The CA membrane layer 22 serves as an interference eliminating
membrane as it is adapted to allow H.sub.2 O.sub.2 to pass through,
but not oxidable agents such as paracetamol, HEPES, and ascorbic
acid which would otherwise interfere with the measuring result.
The CA membrane layer 22 with the applied glucose oxidase layer 23
is provided in a known manner, e.g. as disclosed in the
specification of U.S. Pat. No. 3,979,274, The Yellow Springs
Instrument Company, Inc.
FIG. 2 shows a schematic view of a section of the PETP membrane
layer 24 prior to its surface modification. The section is shown at
one of the pores 25 of the membrane layer 24. For clarity, the
section is somewhat distorted. As seen, the membrane layer 24 has
free --COOH and --OH groups 28 and 29, respectively, on its
surface. The groups are present on the outer surfaces 26, 27 of the
membrane layer as well as on the surfaces of the pores 25.
FIG. 3 shows a schematic view corresponding to the one of FIG. 2,
but showing the PETP membrane layer 24 after surface modification.
As seen, chains 30 of polyethylene glycol are immobilised to the
otherwise free --COOH groups (28 of FIG. 2) of the membrane layer.
The lengths of the chains 30 are adapted so that they, when the
measuring surface of the sensor 2 contacts the blood sample, are
"dissolved" in the aqueous environment and by means of their
"eelgrass"-like movements may prevent macromolecules, such as e.g.
blood proteins, from getting into contact with and coating the
surface of the membrane layer 24.
EXAMPLE 1
Preparation of a Modified PETP Membrane Layer for a Glucose
Sensor
The PETP membrane layer is prepared from a biaxially stretched PETP
foil (Mylar A from Whatman S. A., Louvain La-Neuve, Belgium) of the
following specifications:
Thickness: 12 .mu.m.+-.1 .mu.m
Pore density: 1.6.times.10.sup.6 pores/cm.sup.2 (nominel)
Pore size: approx. 0.1 .mu.m (nominel)
Air flow: 1.6 mL/min/cm.sup.2 at 0.7 Bar (10 psi)
PETP is selected as membrane material particularly on the basis of
requirements to good resistance and strength. The PETP material is
surface modified by a solution of a hydrophilic component of the
following composition:
10 g PEG-200-(OH).sub.2
2 g CMC-MTS
0.2 g Triton CF-54
4000 ml demin. H.sub.2 O
pH 6.0-6.5
where
PEG-200-(OH).sub.2 : Polyethylene glycol with two OH groups, mean
molecular weight 200 g/mol, minimum content of tetraethylene glycol
of 20% and of tri-, tetra- and pentaethylene glycol of 60%.
CMC-MTS:
1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluene
sulphonate, 95% (coupling reagent)
The PETP material is cut into sheets and each sheet is secured to a
frame. The frame with the PETP sheet is dipped into the reaction
bath containing the hydrophilic component for approx. 18 hours at
room temperature. Subsequently, the material is washed while
stirred for approx. 15 minutes at room temperature with 0.1% Triton
X-100 in demineralised water and washed twice while stirred in
demineralised water, each time for approx. 10 minutes. Finally, the
material is dried for at least 16 hours in a ventilated fume
cupboard at room temperature. During this modification chains of
PEG-200 are immobilised to the surface of the PETP material,
including the pore surface, by means of ester binding to the free
--COOH groups on the surface.
The surface modified PETP material is cut in the shape of wafers
which are collected with the previously prepared CA/glucose oxidase
membrane layers 22, 23. The membrane 21 is now ready for further
mounting into the membrane ring 3.
A membrane prepared by means of the above-mentioned procedure has
shown particularly good durability--it is durable for at least 30
days when placed in an operating analyzer.
The PEG-200 chains on the membrane surface serve as a hydrogel,
thus making the surface non-sticky; at the same time being polar
and non-reactive, thus eliminating the capability of i.a. the blood
proteins to form deposits onto the surface.
EXAMPLE 2
Selection of a Hydrophilic Component
During the process of finding a PEG providing the best modified
membrane as to stable measurements, PETP membranes modified with
different types of polyethylene glycol (PEG) (primarily of
different molecular weights) and by slightly different procedures
were prepared. Measurement results from 10 measurements using each
of said membranes on samples of whole blood were compared with each
other and with measurement results from an unmodified membrane and
a membrane washed in MeOH. The results appear from Table 1
below.
TABLE 1 ______________________________________ Decrease during 10
blood Modification measurements %
______________________________________ Unmodified PETP membrane 30
PETP membrane wased in MeOH 7 PEG-5000-NH2 in THF/DCC 7.2
PEG-4000-(COOH).sub.2 8.3 PEG-2000-(COOH).sub.2 4.0
PEG-200-(OH).sub.2, CMC-MTS, aqueous 1.8 PEG-200-(OH).sub.2,
without coupling 5.2 reagent, aqueous PEG-200-OMe, CMC-MTS, aqueous
4.7 ______________________________________
As seen from the table, it is clear that the PETP membrane modified
with PEG-200-(OH).sub.2 according to the above procedure produced
the absolutely best result. The more hydrophobic PEG-200-OMe, with
only one OH group, showed a stronger tendency towards coating of
the membrane surface. Further, there seems to be a tendency towards
a larger decrease of the measurement results with a larger
molecular weight of PEG.
No experiments have been performed with PEG having molecular
weights in the range of 200 g/mol and 2000 g/mol, but it is
assessed that PEG with molecular weights slightly above 200 g/mol,
perhaps up to 1000 g/mol, will be suitable for modification of the
PETP membrane. Further, it is assessed that also PEG with molecular
weights of less than 200 g/mol, e.g. 100 g/mol, will be
suitable.
* * * * *